A wide variety of analytical chemical techniques measure a sample's absorption of radiation at a particular wavelength or series of wavelengths. Often times, the absorption of this specified radiation will vary in a predefined relationship with respect to some specific chemical or physical property of the sample, such as density or concentration. Accordingly, by measuring the absorption of a sample at the specified wavelength or wavelengths, one can frequently determine the density or concentration of some component of the sample.
For example, when radiant energy passes through a liquid, certain wavelengths of that energy may be selectively absorbed by particles which are dissolved in that liquid. For a given path length which the light traverses through the liquid, Beer's law (also referred to as the Beer-Lambert relationship) indicates that the relative transmittance of the liquid at a given wavelength is inversely logarithmically related to the concentration of the solute which absorbs that wavelength. Accordingly, for a sample having a predetermined path length, the transmittance of a sample at the specified wavelength should permit one to fairly readily determine the concentration of the solute which absorbs at that wavelength.
This principle is commonly used in hemoglobinometers, which are essentially specialized spectrophotometers used to measure the concentration of hemoglobin in a sample. By directing a light at a specified wavelength or series of wavelengths into a sample of a known thickness and measuring the intensity of the light passing through the sample, one can effectively determine the concentration of one or more species of hemoglobin in the sample. Such a process is discussed in U.S. Pat. No. 4,357,105 (Loretz) and in U.S. Pat. No. 3,994,585 (Frey), the teachings of both of which are incorporated herein by reference.
The utilization of transmittance as a measure of concentration can provide fairly accurate results in a simple, efficient manner. Unfortunately, this measurement is subject to a number of variables. Some of these variables are dependent on the measuring device itself, such as the nature of the light being emitted by the light source, the spectral response of the other optical components interposed between the light source and the detector, temperature within the system, etc. By utilizing appropriate controls and frequent calibration, such variables can be effectively determined and factored out of any transmittance measurements.
There are some variables which are sample-dependent, though. One such variable which can present significant difficulties in measuring the light attenuation due to absorption of the sample is the presence of scattering particles. If a sample is non-scattering, the total transmittance measured for the sample can provide an accurate measurement of the absorption attributable to the presence of the solute of interest. However, if the sample also scatters the wavelength of radiation of interest, this scattering can significantly impact the measured transmittance of the sample and yield inaccurate analytical results.
The losses attributable to scattering have two primary components. The first is the radiation scattered away from the detector which will never reach the detector at all. The other component is related to the fact that the scattering particles will significantly increase the mean path length of radiation passing through the sample as the radiation bounces from one scattering particle to another on the way to the detector. Since Beer's law is based on an assumption that the path length through the fluid will remain constant, such an increase in the mean path length can have a marked impact on the calculated concentration of the solute.
FIG. 10 illustrates the impact of scattering in a blood sample. Whole blood is made up primarily of plasma and red blood cells, which tend to scatter light. (White blood cells and platelets play a minor role due to the quantity involved.) The presence of the red blood cells, therefore, can have a significant impact on the measured optical density (i.e., the negative of the logarithm of the transmittance value). For this reason, many of the more accurate blood analyzers mechanically or chemically lyse the sample, i.e. break down the cell walls of the red blood cells, before taking any measurements. Since it is the change in the index of refraction of the sample at the surface of the red blood cells that causes scattering, lysing will allow one to achieve a virtually non-scattering sample.
Unfortunately, lysing a sample adds its own complexities. Of one mechanically lyses the sample, this is commonly done in a length of flexible tubing through which each sample must pass. This significantly increases the risk of cross-contamination between the samples. If one chemically lyses the sample, this will dilute the original sample and can make it more difficult to detect smaller hemoglobin concentrations. On addition, lysing will not remove all scattering particles. Sometimes blood includes a not insignificant amount of other light-scattering particles, such as fat particles, and certain drugs, such as one sold under the trade name Interlipid, can also affect scattering. In addition, if lysis is incomplete, the non-lysed cells will continue to scatter light.
FIG. 10 schematically illustrates the relationship between the optical density of a sample and the total hemoglobin concentration THb!. There are two curves depicted in FIG. 10. The lower curve, shown in dashed lines, is the optical density for a lysed blood sample. The slope of this line is constant since the only impact on optical density is the hemoglobin concentration. It should be noted that this graph is somewhat idealized in that a variety of other factors could impact the optical density, as noted above, but those factors are ignored in FIG. 10.
The upper curve, shown in solid lines, is the optical density for a whole blood sample. The slope of this line varies depending on the hemoglobin concentration. Hemoglobin is retained within red blood cells. Generally speaking, therefore, the higher the total hemoglobin in the blood sample, the higher the number of light-scattering red blood cells there will be. As noted above, light-scattering particles will significantly reduce the transmittance, increasing the measured optical density. For this reason, the scattering curve is positioned above the lysed curve along most of its length in FIG. 10. The difference in the measured optical density at any given concentration is indicated as an offset S.
The value of this offset S will differ depending on the concentration of the red blood cells in the sample. Between two end points, indicated as A and B, in FIG. 10, this scattering offset S will remain substantially constant and the slope of the two curves will remain substantially identical. On either end of this range, though, this offset will vary.
In any scattering sample, the offset S will depend on the relative indices of refraction of the scattering particles and the medium in which they are suspended. In the case of whole blood, the plasma, red blood cell walls and the liquid within those cell walls each have different refractive indices. This causes light to bend as it passes from plasma, through the cell wall to the intracellular fluid, and back out again. In addition, since the different refractive indices of the various materials means that the light passes through those materials at different rates, these differences in the indices of refraction will affect the effective light path length through the sample.
Coherent light sources produce light rays with a fixed phase relationship with one another. When the light rays are in phase with one another, their wave maxima will constructively combine to produce a higher total light intensity. If a sample is non-scattering, the path length through the sample is the same for each ray of light, so the phase relationship between the rays remains the same when light passes through the sample. As a result, the rays exiting the sample remain coherent and the wave maxima of the light rays constructively combine.
If the sample is scattering, the path length for each ray is different due to differences in the media through which the light must pass to traverse the sample. As a result, the original phase relationship of the light rays is lost and at least some of the light rays may destructively combine. As the particle concentration of the sample increases, the phase relationship between the light rays is increasingly lost until the phase relationship between any two rays of light exiting the sample is essentially completely random. At this point, the light exiting the sample is said to be "incoherent" and the sample is said to be incoherently scattering.
As the sample becomes increasingly scattering, the light goes from being completely coherent (as in the case of Hct=0 in FIG. 10) to progressively increasing incoherence. As a result, the offset S in FIG. 10 progressively increases from an initial value of zero as the THb value (which is related to the number of light scattering red blood cells, as noted above) increases. Once the light exiting the sample becomes incoherent, though, the offset attributable to such losses will remain fairly constant.
The scattering which occurs at lower particle densities is referred to as "coherent" scattering. "Incoherent" scattering, which occurs at higher particle densities, produces a substantially constant scattering loss over a fairly wide range of particle concentrations due to the essentially random nature of the interaction with the particles. At even higher particle concentrations, the sample begins to scatter coherently again. In essence, the sample can be viewed as scattering particles with a liquid interspersed between those particles, which induces behavior similar to a liquid with particles dispersed throughout the liquid.
Generally, one would expect the sample to be essentially completely incoherently scattering at a hematocrit fraction (Hct) of about 0.25 at the lower end, which will generally correspond to a point where A in FIG. 10 is about 8 g/dL. In a healthy individual, the hemoglobin concentration of the blood will tend to be about 16 g/dL for male adults and about 15 g/dL for female adults. Accordingly, most samples will fall within the incoherent scattering range and have a fixed, predictable increase in optical density attributable to scattering, shown in FIG. 10 as the offset S. However, it is not particularly unusual to have patient samples with hemoglobin concentrations significantly above or below this norm. Some of those samples will fall in the coherent scattering region, which will have a lower, less readily predictable offset S.
Many whole blood hemoglobinometers described in the literature do not take into account the possibility that a patient's hemoglobin concentrations could fall within the coherently scattering ranges. Instead, a fixed offset S is subtracted from every optical density measurement before calculating the hemoglobin concentration. Obviously, the more one deviates from incoherent scattering (e.g., the lower the concentration below the level A in FIG. 10), the more inaccurate the calculated hemoglobin concentration will be. Unfortunately, with current mechanisms, there is no way to determine the transmission losses attributable to scattering on a sample-by-sample basis. Accordingly, if a physician needs to accurately determine lower hemoglobin concentrations, the patient's sample must be analyzed in a different analyzer which will lyse the sample.